Antimicrob Agents Chemother. 2015 Feb;59(2):1088-99. doi: 10.1128/AAC.03659-14.
Antiviral potential of ERK/MAPK and PI3K/AKT/mTOR signaling modulation for Middle East respiratory syndrome coronavirus infection as identified by temporal kinome analysis
Jason Kindrachuk 1, Britini Ork 2, Brit J Hart 2, Steven Mazur 2, Michael R Holbrook 2, Matthew B Frieman 3, Dawn Traynor 2, Reed F Johnson 4, Julie Dyall 2, Jens H Kuhn 2, Gene G Olinger 2, Lisa E Hensley 2, Peter B Jahrling 5
Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus, and infections with this virus can result in acute respiratory syndrome with renal failure. Globally, MERS-CoV has been responsible for 877 laboratory-confirmed infections, including 317 deaths, since September 2012. As there is a paucity of information regarding the molecular pathogenesis associated with this virus or the identities of novel antiviral drug targets, we performed temporal kinome analysis on human hepatocytes infected with the Erasmus isolate of MERS-CoV with peptide kinome arrays. bioinformatics analysis of our kinome data, including pathway overrepresentation analysis (ORA) and functional network analysis, suggested that extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K)/serine-threonine kinase (AKT)/mammalian target of rapamycin (mTOR) signaling responses were specifically modulated in response to MERS-CoV infection in vitro throughout the course of infection. The overrepresentation of specific intermediates within these pathways determined by pathway and functional network analysis of our kinome data correlated with similar patterns of phosphorylation determined through Western blot array analysis. In addition, analysis of the effects of specific kinase inhibitors on MERS-CoV infection in tissue culture models confirmed these cellular response observations. Further, we have demonstrated that a subset of licensed kinase inhibitors targeting the ERK/MAPK and PI3K/AKT/mTOR pathways significantly inhibited MERS-CoV replication in vitro whether they were added before or after viral infection. Taken together, our data suggest that ERK/MAPK and PI3K/AKT/mTOR signaling responses play important roles in MERS-CoV infection and may represent novel drug targets for therapeutic intervention strategies.
Preprint from medRxiv, 03 Sep 2020. DOI: 10.1101/2020.09.02.20186783 PPR: PPR208585
PI3K/mTOR and topoisomerase inhibitors with potential activity against SARS-CoV-2 infection
White JR, Foote MB, Jee J, Argilés G, Wan JC, Diaz LA
There is an urgent need to identify therapies to prevent and treat SARS-CoV-2 infection. We performed a statistical evaluation of in vitro gene expression profiles reflecting exposure to 1,835 drugs, and found topoisomerase inhibitors and PI3K/mTOR pathway inhibitors among the strongest candidates for reduced expression of ACE2, a host gene associated with SARS-CoV-2 infection. Retrospective clinical data suggest that patients on these agents may be less likely to test positive for SARS-CoV-2.
Arch Virol. 2021 Jan 18. doi: 10.1007/s00705-021-04958-7. Online ahead of print.
The roles of signaling pathways in SARS-CoV-2 infection; lessons learned from SARS-CoV and MERS-CoV
Nima Hemmat 1, Zahra Asadzadeh # 1, Noora Karim Ahangar # 1, Hajar Alemohammad # 1, Basira Najafzadeh # 1, Afshin Derakhshani 1 2, Amir Baghbanzadeh 1, Hossein Bannazadeh Baghi 1 3 4, Darya Javadrashid 1, Souzan Najafi 1, Meriadeg Ar Gouilh 5 6, Behzad Baradaran 7 8
The number of descriptions of emerging viruses has grown at an unprecedented rate since the beginning of the 21st century. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), is the third highly pathogenic coronavirus that has introduced itself into the human population in the current era, after SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Molecular and cellular studies of the pathogenesis of this novel coronavirus are still in the early stages of research; however, based on similarities of SARS-CoV-2 to other coronaviruses, it can be hypothesized that the NF-κB, cytokine regulation, ERK, and TNF-α signaling pathways are the likely causes of inflammation at the onset of COVID-19. Several drugs have been prescribed and used to alleviate the adverse effects of these inflammatory cellular signaling pathways, and these might be beneficial for developing novel therapeutic modalities against COVID-19. In this review, we briefly summarize alterations of cellular signaling pathways that are associated with coronavirus infection, particularly SARS-CoV and MERS-CoV, and tabulate the therapeutic agents that are currently approved for treating other human diseases.
J Mol Cell Cardiol. 2020 Jul; 144: 63–65.
p38 MAPK inhibition: A promising therapeutic approach for COVID-19
Joseph M. Grimesa and Kevin V. Grimesb,⁎
COVID-19, caused by the SARS-CoV-2 virus, is a major source of morbidity and mortality due to its inflammatory effects in the lungs and heart. The p38 MAPK pathway plays a crucial role in the release of pro-inflammatory cytokines such as IL-6 and has been implicated in acute lung injury and myocardial dysfunction. The overwhelming inflammatory response in COVID-19 infection may be caused by disproportionately upregulated p38 activity, explained by two mechanisms. First, angiotensin-converting enzyme 2 (ACE2) activity is lost during SARS-CoV-2 viral entry. ACE2 is highly expressed in the lungs and heart and converts Angiotensin II into Angiotensin 1–7. Angiotensin II signals proinflammatory, pro-vasoconstrictive, pro-thrombotic activity through p38 MAPK activation, which is countered by Angiotensin 1–7 downregulation of p38 activity. Loss of ACE2 upon viral entry may tip the balance towards destructive p38 signaling through Angiotensin II. Second, SARS-CoV was previously shown to directly upregulate p38 activity via a viral protein, similar to other RNA respiratory viruses that may hijack p38 activity to promote replication. Given the homology between SARS-CoV and SARS-CoV-2, the latter may employ a similar mechanism. Thus, SARS-CoV-2 may induce overwhelming inflammation by directly activating p38 and downregulating a key inhibitory pathway, while simultaneously taking advantage of p38 activity to replicate. Therapeutic inhibition of p38 could therefore attenuate COVID-19 infection. Interestingly, a prior preclinical study showed protective effects of p38 inhibition in a SARS-CoV mouse model. A number of p38 inhibitors are in the clinical stage and should be considered for clinical trials in serious COVID-19 infection.
SARS-CoV-2 and the Possible Role of Raf/MEK/ERK Pathway in Viral Survival: Is This a Potential Therapeutic Strategy for COVID-19?
Ghasemnejad-Berenji M.a · Pashapour S.b
In late 2019, a sudden rise in respiratory-related disease cases in China triggered the identification of its source as a novel corona virus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease caused by novel SARS-CoV-2 is named as COVID-19 by the World Health Organization . The culprit virus belongs to the Coronaviridae family of coronaviruses that caused 2 other outbreaks, namely, severe acute respiratory syndrome (SARS) in 2002  and Middle East respiratory syndrome in 2012 . As the survival of every virus depends on its host cell, the understanding of cellular functions such as signaling pathways that are essential for viral replication may be suitable to define targets for antiviral therapy and pave the way toward effective drugs against essential cellular activities supporting viral replication . In this regard, we have focused on the Raf/MEK/ERK signaling pathway, which is probably one of the most well-known signal transduction pathways among biologists because of its implication in a wide variety of cellular functions such as cell proliferation, cell cycle arrest, and apoptosis . The mechanism of this pathway is initiated by G protein-coupled receptors, which leads to the phosphorylation of downstream molecules and activates the serine threonine kinase Raf (dual specificity kinase MEK and MAPK/ERK). ERK phosphorylates various substrates, transforms the signals, and follows different functions in cells . Hence, it is not surprising that several DNA and RNA viruses inherit this pathway, apart from an initial activation upon viral attachment, for various steps in the viral life cycle . Consequently, the kinetic of pathway activation is highly dynamic . For example, herpes simplex type-1 virus-induced activation of the Raf/MEK/ERK pathway is used for cytoskeleton rearrangement during entry , JC polyomavirus requires ERK activation for viral transcription , and influenza A virus hijacks Raf/MEK/ERK activity for efficient viral ribonucleoprotein export [11, 12]. Ebola virus glycoprotein-induced cytotoxicity depends on ERK activation , and hepatitis C virus relies on Raf/MEK/ERK-mediated upregulation of cytosolic phospholipase A2 for efficient particle production . Flaviviruses, including yellow fever virus, Saint-Louis encephalitis virus, and dengue virus, as well depend on Raf/MEK/ERK-mediated signaling for efficient replication [15, 16]. It has been shown that activated ERK1/2 enhanced the infectivity of human immunodeficiency virus, whereas treatment of cells with the MEK1/2 inhibitor PD98059 significantly inhibited human immunodeficiency virus infectivity [17, 18]. Treating cells with UO126, a highly selective inhibitor of both MEK1 and MEK2, also significantly inhibited the propagation of influenza A virus, Borna disease virus, coxsackievirus B3, and HCMV [11, 19-21]. Thus, it appears that the MEK1/2 inhibitors have a broad effect on propagations of viruses from various families (positive-strand and negative-strand RNA viruses, retroviruses, and DNA viruses) . SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA beta-coronavirus. Similar to SARS and Middle East respiratory syndrome, the SARS-CoV-2 genome encodes nonstructural proteins (such as 3-chymotrypsin-like protease, papain-like protease, helicase, and RNA-dependent RNA polymerase), structural proteins (such as spike glycoprotein), and accessory proteins. The 4 nonstructural proteins mentioned above are key enzymes in the viral life cycle, and the spike glycoprotein is indispensable for virus-cell receptor interactions during viral entry . By analyzing the effects of transiently expressed viral spike protein (S) of SARS-CoV, it was revealed that the S protein plays an important role in virus-stimulated cyclooxygenase-2 (COX-2) expression . COX-2 is a prostaglandin synthetase involved in inflammation  that is highly regulated by different factors including cytokines . The upstream calcium-dependent PKCa that modulates the downstream Raf/MEK/ERK pathway is induced by the SARS-CoV S protein. It was revealed that ERK is involved in S protein-induced activation of the COX-2 promoter and the production of COX-2 protein in HEK293T cells. This result helps explain the function of SARS-CoV S protein in SARS pathogenesis . More information on relevant MAPK signaling induced by coronavirus was obtained for murine coronavirus. It was revealed that infection of cultured cells with murine coronavirus resulted in activation of the Raf/MEK/ERK signal cascade, and inhibition of the MAPK signaling pathway by U0126 or knockdown of MEK and ERK by small interfering RNAs significantly impaired murine coronavirus progeny production. The treatment did not affect virus entry or cellular and viral mRNA production. However, synthesis of viral genomic and sub-genomic RNAs was severely suppressed by U0126 treatment. This study indicated that the MAPK signaling pathway is involved in murine coronavirus RNA synthesis . Selective RAF inhibitors are well tolerated, and severe toxicities occur infrequently in noninfected cells. Among the common grade 1–2 adverse events are dermatological affections (50–70%), fatigue (30–50%), diarrhea (10–30%), and nausea (10–20%) . Considering the role of the Raf/MEK/ERK signaling pathway in the pathogenesis of various viruses, it is probable that the activation of this signaling pathway by COVID-19 has an important role in the survival of this virus. In this regard, drugs that inhibit the Raf/MEK/ERK signaling pathway may be potential antiviral candidates for the treatment of COVID-19.
Antiviral Res. 2016 Sep;133:140-4. doi: 10.1016/j.antiviral.2016.08.003. Epub 2016 Aug 4.
A Raf kinase inhibitor demonstrates antiviral activities both in vitro and in vivo against different genotypes of virulent Newcastle disease virus
Renfu Yin 1, Xinxin Liu 2, Pingze Zhang 3, Yanyu Chen 3, Guangyao Xie 3, Lili Ai 3, Cong Xue 3, Jing Qian 3, Yuhai Bi 4, Jianjun Chen 5, Yuzhang Sun 6, Tobias Stoeger 7, Zhuang Ding 8
Newcastle disease (ND) is still one of the major plagues of birds worldwide. Combat actions are limited to vaccines, highlighting the urgent need for new and amply available antiviral drugs. Previous results have shown that Newcastle disease virus (NDV) downregulates the intracellular Raf kinase inhibitor protein (RKIP) expression for efficient replication, suggesting that this molecular may be a suitable target for antiviral intervention. In the present work, we investigated whether or not the Raf kinase inhibitor V (RKIV), which functions in the same way as RKIP by targeting the intracellular Raf kinase, is able to suppress the propagation of enzootic virulent NDV in vitro and in vivo. In vitro antiviral activity of RKIV was assessed by cell-based assay, and in vivo activity was determined in the chicken model. Our results clearly showed that RKIV treatment protected the cells from NDV-induced CPE with the effective concentrations on nM level, and inhibited virus replication in the lungs of infected chickens in a dose-dependent manner and protected chickens from the lethal infection by NDV. Thus, we conclude that the Raf kinase inhibitor compound RKIV, by inhibiting the host cellular target Raf kinase, might be very promising as a new class of antivirals against the enzootic virulent NDV infection.
Front Microbiol. 2017; 8: 2426.
Vemurafenib Limits Influenza A Virus Propagation by Targeting Multiple Signaling Pathways
Magdalena Holzberg,1,† Yvonne Boergeling,1,2,† Tobias Schräder,1 Stephan Ludwig,1,2 and Christina Ehrhardt1,2,*
Influenza A viruses (IAV) can cause severe global pandemic outbreaks. The currently licensed antiviral drugs are not very effective and prone to viral resistance. Thus, novel effective and broadly active drugs are urgently needed. We have identified the cellular Raf/MEK/ERK signaling cascade as crucial for IAV replication and suitable target for an antiviral intervention. Since this signaling cascade is aberrantly activated in many human cancers, several clinically approved inhibitors of Raf and MEK are now available. Here we explored the anti-IAV action of the licensed B-RafV600E inhibitor Vemurafenib. Treatment of B-RafWT cells with Vemurafenib induced a hyperactivation of the Raf/MEK/ERK cascade rather than inhibiting its activation upon IAV infection. Despite this hyperactivation, which has also been confirmed by others, Vemurafenib still strongly limited IAV-induced activation of other signaling cascades especially of p38 and JNK mitogen-activated protein kinase (MAPK) pathways. Most interestingly, Vemurafenib inhibited virus-induced apoptosis via impaired expression of apoptosis-inducing cytokines and led to hampered viral protein expression most likely due to the decreased activation of p38 and JNK MAPK. These multiple actions resulted in a profound and broadly active inhibition of viral replication, up to a titer reduction of three orders of a magnitude. Thus, while Vemurafenib did not act similar to MEK inhibitors, it displays strong antiviral properties via a distinct and multi-target mode of action.
Virology. 2015 Sep;483:126-40. doi: 10.1016/j.virol.2015.04.017. Epub 2015 May 15.
An unexpected inhibition of antiviral signaling by virus-encoded tumor suppressor p53 in pancreatic cancer cells
Eric Hastie 1, Marcela Cataldi 1, Nury Steuerwald 2, Valery Z Grdzelishvili 3
Virus-encoded tumor suppressor p53 transgene expression has been successfully used in vesicular stomatitis virus (VSV) and other oncolytic viruses (OVs) to enhance their anticancer activities. However, p53 is also known to inhibit virus replication via enhanced type I interferon (IFN) antiviral responses. To examine whether p53 transgenes enhance antiviral signaling in human pancreatic ductal adenocarcinoma (PDAC) cells, we engineered novel VSV recombinants encoding human p53 or the previously described chimeric p53-CC, which contains the coiled-coil (CC) domain from breakpoint cluster region (BCR) protein and evades the dominant-negative activities of endogenously expressed mutant p53. Contrary to an expected enhancement of antiviral signaling by p53, our global analysis of gene expression in PDAC cells showed that both p53 and p53-CC dramatically inhibited type I IFN responses. Our data suggest that this occurs through p53-mediated inhibition of the NF-κB pathway. Importantly, VSV-encoded p53 or p53-CC did not inhibit antiviral signaling in non-malignant human pancreatic ductal cells, which retained their resistance to all tested VSV recombinants. To the best of our knowledge, this is the first report of p53-mediated inhibition of antiviral signaling, and it suggests that OV-encoded p53 can simultaneously produce anticancer activities while assisting, rather than inhibiting, virus replication in cancer cells.
Am J Physiol Lung Cell Mol Physiol. 2020 Jul 1; 319(1): L45–L47.
Is targeting Akt a viable option to treat advanced-stage COVID-19 patients?
Payaningal R. Somanathcorresponding author1,2,3
One of the primary reasons for high mortality in the advanced-stage coronavirus disease-2019 (COVID-19) patients is the uncontrolled inflammation in the lungs leading to acute respiratory distress syndrome (ARDS). Controlling the pathological inflammation in the ARDS lungs without compromising the immune system’s fight against the virus is indeed a daunting task. In this situation, an appropriate therapeutic target would be the one that will not only reverse the damaging inflammation and promote resolution but also helps to check the root cause of the virus infection. Akt is a potential therapeutic target for the advanced-stage COVID-19 patients; its inhibition will potentially suppress the pathological inflammation, cytokine storm, fibroproliferation, and platelet activation associated with COVID-19, and at the same time prevent scarring and promote resolution in injured lungs. As pharmacological inhibition of Akt has also been reported to inhibit angiotensin-converting enzyme 2 (ACE2) expression, a receptor for the virus entry into the lung cells, targeting Akt for COVID-19 looks a viable option.
Severe acute respiratory syndrome (SARS), an acute respiratory distress syndrome (ARDS) caused by the coronavirus-2 (SARS-CoV-2), is the primary reason for high mortality associated with coronavirus disease-2019 (COVID-19) (25). The prolonged asymptomatic incubation period in COVID-19 patients ranging between 1 and 14 days (5.1 days median) is a major bottleneck in its early detection and preventing it from infecting the lungs of some patients (12). Additionally, comorbidities such as diabetes and hypertension (10, 24) and a history of medications such as angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme (ACE) inhibitors (9) further worsens the COVID-19 associated ARDS. The effect of comorbidities (14) and the use of cardiovascular medications (19) are believed to be due to the increased expression of ACE2, a putative receptor for SARS-CoV-2 (23). As current treatment options for ARDS are very limited (21, 22), treating advanced COVID-19 patients is a bigger challenge due to the reasons above.
Early detection and preventing a lung infection would be the most desirable approach to treat COVID-19 patients. Although some COVID-19 patients present early symptoms, many patients harboring SARS-CoV-2 remain on a long asymptomatic period and are undetected until complications arise (10, 12). Having a large population suffering from metabolic diseases and hypertension in Western countries (17), the potential risk of COVID-19 associated ARDS is also higher. Hence, it is not always possible to cure COVID-19 patients before severe lung infection takes hold. The next obvious question is, how do we treat advanced-stage COVID-19 patients with lung infections? One potential option would be to target ACE2 or pathways promoting ACE2 expression or activation. Alternately, COVID-19 patients can also be treated by engineered peptides, antibodies, or compounds to disrupt or prevent the interaction between SARS-CoV-2 spike protein and ACE2 (23) that will prevent reinfections and limit the lung damage. A better option to manage advanced-stage COVID-19 patients would be a drug that can not only suppress inflammation but also promote the resolution of lung injury and prevent scarring (Fig. 1).
Akt inhibition will increase the number of CD4+/FoxP3+/CD103+/CTLA4+ effector regulatory T cells (Tregs) in the acute respiratory distress syndrome (ARDS) lung to suppress inflammation and promote injury resolution. In the ARDS lung, reduced Akt activity in conventional T cells will promote their differentiation to effector Tregs, limiting inflammation and scar formation, and promoting vascular regeneration and wound resolution, revealing the potential therapeutic benefits of Akt inhibitors such as triciribine and MK2206 to treat ARDS in advanced-stage coronavirus disease-2019 (COVID-19) patients.
The phosphoinositide 3 (PI3)-kinase/Akt pathway promotes inflammation in several disease states (8). Whereas genetic deletion of Akt1 gene reduced inflammation and improved cardiac function in mice following myocardial ischemia (13, 16), pharmacological Akt inhibition suppressed inflammation in mice, myofibroblast differentiation, and prevented vascular rarefaction to halt pulmonary fibrosis progression (1, 2). Furthermore, whereas Akt1 deficiency in macrophages resulted in reduced foam cell formation (13), inhibition of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) leading to Akt activation in the regulatory T cells (Tregs) promoted inflammation (18). Adoptive transfer of Tregs has demonstrated to suppress fibroproliferation and improve injury resolution in an animal model of experimental lung injury (6, 15), suggesting that increasing the number of Tregs in ARDS lungs would be an ideal strategy to treat COVID-19 patients in the advanced stages. However, the pharmacological means to increase the number of Tregs in ARDS lung was not available until we recently demonstrated that the number of the effector (activated) Tregs in the advanced stages of bacterial endotoxin-induced experimental lung injury in mouse lungs can be increased by Akt inhibition with compounds such as triciribine and MK2206, promoting injury resolution and recovery (3). Whereas ACE2 has been implicated in pulmonary arterial hypertension (7) and lung inflammation (11), Akt inhibition with MK2206 and triciribine has been reported to ameliorate the pathological effects of ACE2 in hepatic steatosis (4). However, a link between the Akt pathway and ACE2 activation in COVID-19 patients needs to be investigated.
Although the host PI3-kinase/Akt pathway is utilized by the viruses in general for its survival and replication (5, 20), this has not been demonstrated in the case of the SARS-CoV-2 virus replication in the lung epithelial cells. On the contrary, an adverse effect of Akt suppression by promoting the SARS-CoV-2 virus replication in the patient lung epithelial cells also cannot be ruled out. Nevertheless, pharmacological inhibition of the Akt pathway using inhibitors such as triciribine and MK2206, alone or in combination with the currently evolving standard of care, provides potential treatment options for COVID-19 patients with ARDS. Based on the preclinical observations from non-COVID-19 lung disease research, Akt suppression is expected to increase Tregs in the lungs of COVID-19 patients, in turn, suppressing inflammation and fibroproliferation, promoting the resolution of injury, and preventing vascular pruning in the lungs as a result of SARS-CoV-2 infection. This, however, needs further experimental validation in a suitable preclinical COVID-19 model such as the non-human primates before clinical trials are conducted in COVID-19 patients.
Virology. 2004 Oct 1;327(2):169-74. doi: 10.1016/j.virol.2004.07.005.
Importance of Akt signaling pathway for apoptosis in SARS-CoV-infected Vero E6 cells
Tetsuya Mizutani 1, Shuetsu Fukushi, Masayuki Saijo, Ichiro Kurane, Shigeru Morikawa
Severe acute respiratory syndrome (SARS) is an acute respiratory tract infectious disease that is associated with a new coronavirus (SARS-CoV). Our recent study indicated that SARS-CoV infection induces activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway and the p38 MAPK inhibitor partially inhibited its cytopathic effect in Vero E6 cells. The results of the present study indicated that before cell death, Akt, which is an inhibitor of apoptosis, was also activated in response to viral replication. Phosphorylation of a serine residue on Akt was detected at least 8 h postinfection (hpi), which declined after 18 hpi. Thus, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in virus-infected Vero E6 cells. However, a threonine residue was not phosphorylated. A downstream target of Akt, glycogen synthase kinase 3beta (GSK-3beta), was slightly phosphorylated, indicating that the level of activation of Akt was very low. PKCzeta, which is downstream of the PI3K pathway, was also phosphorylated in virus-infected cells. These results suggested that weak activation of Akt cannot prevent apoptosis induced by SARS-CoV infection in Vero E6 cells.